Approximately one-third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of human tuberculosis. Tuberculosis kills almost three million each year worldwide and has shown an increasing prevalence in the United States over the past few years. Although several antituberculosis drugs are available, they are usually administered in combination for long periods and poor compliance has contributed to the increase in strains of M. tuberculosis that are resistant to these drugs. The recent increase in the proportion of multiple drug resistant strains of M. tuberculosis in the United States and the high mortality of these infections is particularly alarming. There is clearly a clinical need for new effective drugs that recognize different cellular targets, improved diagnostic strategies and development of effective vaccines. Antibiotics act through the inhibition of essential cellular functions. Conditional-lethal mutants of the mycobacteria will be isolated that are conditionally defective in essential gene functions such that they either die or cannot grow at the non-permissive condition. In particular, temperature-sensitive mutants that result from either missense mutations in essential genes, or due to nonsense mutations in essential genes in the context of a temperature-sensitive tRNA suppressor will be characterized. The essential genes identified by the mutations will be cloned by complementation of the conditional-lethal phenotype using cosmid libraries and the genes characterized. Many of the essential genes identified will be potential targets for antimycobacterial agents and will serve as an initiation point for the rationale design of novel antitubercular drugs.